Nitrite binding to globins: linkage isomerism, EPR silence and reductive chemistry

The nitrite adducts of globins can potentially bind via O- or N- linkage to the heme iron. We have used EPR (electron paramagnetic resonance) and DFT (density functional theory) to explore these binding modes to myoglobin and hemoglobin. We demonstrate that the nitrite adducts of both globins have detectable EPR signals; we provide an explanation for the difficulty in detecting these EPR features, based on uniaxial state considerations. The EPR and DFT data show that both nitrite linkage isomers can be present at the same time and that the two isomers are readily interconvertible in solution. The millisecond-scale process of nitrite reduction by Hb is investigated in search of the elusive Fe(II)-nitrite adduct

The Reaction of Oxy Hemoglobin with Nitrite: Mechanism, Antioxidant-Modulated Effect, and Implications for Blood Substitute Evaluation

The autocatalytic reaction between nitrite and the oxy form of globins involves free radicals. For myoglobin (Mb), an initial binding of nitrite to the iron-coordinated oxygen molecule was proposed; the resulting ferrous-peroxynitrate species was not detected, but its decay product, the high-valent ferryl form, was demonstrated in stopped-flow experiments. Reported here are the stopped flow spectra recorded upon mixing oxy Hb (native, as well as chemically-derivatized in the form of several candidates of blood substitutes) with a supraphysiological concentration of nitrite. The data may be fitted to a simple kinetic model involving a transient met-aqua form, in contrast to the ferryl detected in the case of Mb in a similar reaction sequence. These data are in line with a previous observation of a transient accumulation of ferryl Hb under auto-catalytic conditions at much lower concentrations of nitrite (Grubina, R. et al. J. Biol. Chem. 2007, 282, 12916). The simple model for fitting the stopped-flow data leaves a small part of the absorbance changes unaccounted for, unless a fourth species is invoked displaying features similar to the oxy and tentatively assigned as ferrous-peroxynitrate. Density functional theory (DFT) calculations support this latter assignment. The reaction allows for differentiating between the reactivities of various chemically modified hemoglobins, including candidates for blood substitutes. Polymerization of hemoglobin slows the nitrite-induced oxidation, in sharp contrast to oxidative-stress type reactions which are generally accelerated, not inhibited. Sheep hemoglobin is found to be distinctly more resistant to reaction with nitrite compared to bovine Hb, at large nitrite concentrations (stopped-flow experiments directly observing the oxy + nitrite reaction) as well as under auto-catalytic conditions. Copolymerization of Hb with bovine serum albumin (BSA) using glutaraldehyde leads to a distinct increase of the lag time compared to native Hb as well as to any other form of derivatization examined in the present study. The Hb-BSA copolymer also displays a slower initial reaction with nitrite under stopped-flow conditions, compared to native Hb

Achillea schurii Flowers: Chemical, Antioxidant, and Antimicrobial Investigations

This study aims to evaluate the phenolic profile, and antioxidant and antimicrobial activity of Achillea schurii Sch.-Bip., an endemic species from Romania that has not been investigated yet. The chromatographic profile of the phenolic components was obtained using the HPLC-MS method, while the total polyphenol, flavonoid, caffeic acid derivative contents were quantified using spectrophotometric methods. The antioxidant activity was evaluated using different methods: DPPH radical scavenging, hemoglobin ascorbate peroxidase activity inhibition (HAPX), inhibition of lipid peroxidation catalyzed by cytochrome c, and direct detection of plant-derived free radicals using electron paramagnetic resonance (EPR). The antimicrobial test was performed using the disk diffusion assay. The phenolic profile has revealed high amounts of isoquercitrin, rutin, luteolin, and apigenin. The A. schurii extract exhibited a good antioxidant capacity, and high phenolic contents (76.93 mg/g polyphenols, 18.61 mg/g flavonoids and 41.48 mg/g caffeic acid derivatives, respectively). The antimicrobial tests reveal a remarkable inhibitory activity against Listeria monocytogenes, Staphylococcus aureus, and Salmonella typhimurium. Considering the above, A. schurii may be deemed to offer good perspectives for pharmaceutical and industrial applications

Ascorbate peroxidase activity of cytochrome<i>c</i>

The peroxidase-type reactivity of cytochrome c is proposed to play a role in free radical production and/or apoptosis. This study describes cytochrome c catalysis of peroxide consumption by ascorbate. Under conditions where the sixth coordination position at the cytochrome c heme iron becomes more accessible for exogenous ligands (by carboxymethylation, cardiolipin addition or by partial denaturation with guanidinium hydrochloride) this peroxidase activity is enhanced. A reaction intermediate is detected by stopped-flow UV-vis spectroscopy upon reaction of guanidine-treated cytochrome c with peroxide, which resembles the spectrum of globin Compound II species and is thus proposed to be a ferryl species. The ability of physiological levels of ascorbate (10μM) to interact with this species may have implications for mechanisms of cell signalling or damage that are based on cytochrome c/peroxide interactions. © 2011 Informa UK, Ltd

Antimicrobial and Antioxidant Activities and Phenolic Profile of Eucalyptus globulus Labill. and Corymbia ficifolia (F. Muell.) K.D. Hill &amp; L.A.S. Johnson Leaves

This study was performed to evaluate the in vitro antimicrobial and antioxidant activities and the phenolic profile of Eucalytus globulus Labill. and Corymbia ficifolia (F. Muell.) K.D. Hill &amp; L.A.S. Johnson leaves. Both leave extracts contain significant amounts of phenolic compounds, mainly flavonoids. Qualitative and quantitative analyses of the phenolic compounds were performed using a HPLC/MS method. The main flavonoid was hyperoside and its highest amount was found in E. globulus (666.42 ± 5.02 μg/g dw plant material). Regarding the flavonol profile, myricetin was the dominant compound and its highest amount was found in C. ficifolia leaves (124.46 ± 0.24 μg/g dw plant material). The antioxidant activity was evaluated by DPPH, TEAC, hemoglobin ascorbate peroxidase activity inhibition (HAPX) and inhibition of lipid peroxidation catalyzed by cytochrome c assays, revealing an important antioxidant potential for both species. In the antimicrobial assays, C. ficifolia extract was found to be more active than E. globulus against both Gram-positive and Gram-negative bacterial strains with the exception of Bacillus subtilis. The results of the present study provide new valuable data regarding the bioactivities of these medicinal species

Polyphenolic Content, Antioxidant and Antimicrobial Activities of Lycium barbarum L. and Lycium chinense Mill. Leaves

This study was performed to evaluate the in vitro antioxidant and antimicrobial activities and the polyphenolic content of Lycium barbarum L. and L. chinense Mill. leaves. The different leave extracts contain important amounts of flavonoids (43.73 ± 1.43 and 61.65 ± 0.95 mg/g, respectively) and showed relevant antioxidant activity, as witnessed by the quoted methods. Qualitative and quantitative analyses of target phenolic compounds were achieved using a HPLC-UV-MS method. Rutin was the dominant flavonoid in both analysed species, the highest amount being registered for L. chinense. An important amount of chlorogenic acid was determined in L. chinense and L. barbarum extracts, being more than twice as high in L. chinense than in L. barbarum. Gentisic and caffeic acids were identified only in L. barbarum, whereas kaempferol was only detected in L. chinense. The antioxidant activity was evaluated by DPPH, TEAC, hemoglobin ascorbate peroxidase activity inhibition (HAPX) and inhibition of lipid peroxidation catalyzed by cytochrome c assays revealing a better antioxidant activity for the L. chinense extract. Results obtained in the antimicrobial tests revealed that L. chinense extract was more active than L. barbarum against both Gram-positive and Gram-negative bacterial strains. The results suggest that these species are valuable sources of flavonoids with relevant antioxidant and antimicrobial activities

Antioxidant activity evaluation by physiologically relevant assays based on haemoglobin peroxidase activity and cytochrome <i>c</i>-induced oxidation of liposomes

<p>Two new protocols for exploring antioxidant-related chemical composition and reactivity are described: one based on a chronometric variation of a haemoglobin ascorbate peroxidase assay and one based on cytochrome <i>c</i>-induced oxidation of lecithin liposomes. Detailed accounts are given on their design, application, critical correlations with established methods and mechanisms. These assays are proposed to be physiologically relevant and bring new information regarding a real sample, both qualitative and quantitative. The well-known assays used for evaluation of antioxidant (re)activity are revisited and compared with these new methods. Extracts of the <i>Hedera helix</i> L. are examined as test case, with focus on seasonal variation and on leaf, fruit and flower with respect to chromatographic, spectroscopic and reactivity properties. According to the set of assays performed, winter are the most antioxidant, followed by summer leaves, and then by flowers and fruits.</p